Protein-ribonucleic acid (RNA) interactions play a key role in many fundamental life processes In living organisms, these polymers are often found complexed together in extremely large assemblies whose molecular mass may reach several millions of daltons. In the pathway of gene expression one finds transcribing complexes, containing RNA polymerase in action on a DNA template, with associated nascent RNA. Concurrently, the resulting precursor messenger RNA (pre-mRNA) becomes associated with a multitude of proteins and additional small RNA molecules into a large ribonucleoprotein (RNP) complex, the spliceosome, where it is processed to mature mRNA. Protein synthesis then takes place in the cytoplasm on a third class of particles—the ribosomes. In addition, a number of large protein complexes require mononucleotides (e.g., ATP, GTP) for their assembly and/or catalytic activity.
For such inherently polymorphic assemblies, visualization by transmission electron microscopy (TEM) provides structural information at a resolution that is difficult to obtain in any other way (Chiu and Schmid, 1997; Griffith et al., 1997). Yet, localization and tracing by electron microscopy of RNA or ribonucleotides within such large biological assemblies, are not yet a straightforward undertaking. Even when crystals amenable to X-ray crystallography analysis can be obtained, as is the case for ribosomes, there is still a demand for heavy atom derivatives to phase the diffraction data (Weinstein et al, 1992). Covalent derivatization of RNA with heavy atoms should enable visualization of RNA within RNP complexes by EM and ensure the introduction of electron-dense centers at distinct locations within crystallized RNA molecules and RNP complexes.
Visualization of nucleic acid molecules by TEM cannot be directly achieved because of the low-density weakly scattering atoms they contain. Nevertheless, methods such as electron spectroscopic imaging (e.g., Bazett-Jones, 1992), tungsten shadow casting (e.g., Wang et al., 1994), atomic force microscopy (AFM; e.g. Hansma et al., 1996; Smith et al., 1997), and scanning tunneling microscopy (e.g., Guckenberger et al., 1994) have been used to visualize naked RNA and DNA molecules. More recently, a positive staining protocol that allows visualization of nucleic acids (Dubochet et al., 1971) was used to visualize RNA strands emanating from supraspliceosome particles (Muller et al., 1998), yet RNA located within the particles was not visible. Tagging such macromolecules with clusters of heavy atoms should facilitate their visualization by conventional TEM. The present most popular method employs colloidal gold noncovalently attached to specific antibodies, protein A or other macromolecular probes. For example, attempts were made to visualize spliceosomes by dark-field scanning transmission electron microscopy (STEM) after tagging with biotinylated oligonucleotides complementary to the pre-mRNA that had been conjugated to a streptavidin-colloidal gold complex (Sibbald et al., 1993).
The use of probes with covalently conjugated gold compounds provides a number of advantages over colloidal gold. These include better stability, size uniformity, and complete absence of aggregation, all of which result in better sensitivity and resolution. A number of gold clusters containing a core of 11 gold atoms surrounded by a hydrophilic organic shell of aryl-phosphines have been described (Safer et al., 1986). These undecagold compounds have the general formula Au11L6L′X3, where L is tris(4N-methylcarboxamidophenyl)phosphine, and L′ is a similar ligand in which the methylcarboxamido group on one of the benzene rings is replaced by an activatable side chain such as an ω-amino alkyl group. Activation of this compound with a maleimido group yields a gold cluster that can be conveniently coupled to free thiol groups of proteins (Safer et al., 1986; Wenzel and Baumeister, 1995). An interesting example is the specific labeling with undecagold of the ribosomal protein BL11 within the 50S ribosomal subunit of Bacillus stearothermophilus for its subsequent use as a heavy atom derivative for crystallographic studies (Weinstein et al., 1989, 1992). The same authors also labeled tRNAphe of the same organism by taking advantage of the modified nucleoside 3-(3-amino-3-carboxypropyl) uridine at position 47. The exposed primary amine of this base was reacted with 2-iminothiolane to extend the aliphatic chain and introduce a primary thiol group, which was then coupled to maleimido undecagold (Weinstein et al., 1992).
The diameter of the undecagold cluster is 0.82 nm. It can thus be visualized by high-resolution STEM, but not readily by conventional TEM unless the signal is enhanced by silver enhancement (Burry et al., 1992). Visualization by conventional TEM can be improved by using a larger, 1.4 run. gold cluster (Hainfeld and Furuya, 1992). The structure of this reagent, now commercially available from Nanoprobes (Stony Brook, N.Y.) under the trademark “NANOGOLD”. has not yet been reported. It has nevertheless been used successfully to label proteins (Boisset et al., 1992; Hainfeld and Furuya, 1992) as well as the 5′ or 3′ ends of DNA oligonucleotides (Alivisatos et al., 1996).
The present invention teaches a general systematic strategy for incorporating gold clusters into nucleotides and nucleic acid molecules.